Microtome s

1. Microtomes Brian Cheung Bernard Hwang David Yu San Jose State University College of Engineering

2. Background • The earliest form of microtomy was the freehand sectioning of fresh or fixed material using a sharp razor. • The first microtome used were manual sectioning instruments made from wood, that were mainly used in Botany for cutting plants. • Modern microtomes are precision instruments that cut uniformly thin sections of a variety of materials for microscopic examination. San Jose State University College of Engineering

3. Parts of a Microtome San Jose State University College of Engineering

4. San Jose State University College of Engineering

5. Capabilities • Standard microtomes are used to cut paraffin imbedded tissue specimens into thin plane sections. • Sectioned material must be reproducible from only a fraction of a micron to a few microns thick. (Leitz 1512: 1 – 25mm) • Movements must be precisely controlled to ensure quality of the sectioned material. • Thickness • Reproducibility • Plane parallelism San Jose State University College of Engineering

6. Rotary Microtome Operation • Insert paraffin-embedded sample into sample holder and release spring loaded clamp. • Clamp blade into blade holder. San Jose State University College of Engineering

7. Adjust blade holder angle and tighten San Jose State University College of Engineering

8. Adjust thickness of sample section. • Set counter dial to zero. • Turn crank (one revolution per section). San Jose State University College of Engineering

9. microtome pics\MVI_3845.AVI San Jose State University College of Engineering

10. Precision Engineering Applications • Constraint of the Sample Block • Constraint of the Knife Blade • Microtome Knife Material Selection • Translational Guide Rails • Digital Volumetric Imaging San Jose State University College of Engineering

11. Constraint of the Sample Block • Embedded sample is held against a flat surface and clamped down. • Semi-kinematic Constraint With A Line Contact San Jose State University College of Engineering

12. Constraint of the Knife Blade • Knife is placed in a retaining platform and secured rigidly in place to the microtome by preloading the opposite side of the knife. • If disposable knives are used, the knife is first held in a fixture; and the fixture is secured to the microtome. San Jose State University College of Engineering

13. Alternative Constraint Methods • Newer microtomes may use additional methods to secure the blade. • Additional Tightening Screws For Additional Point Contacts • Knife Holder With Magnetic Retaining Points San Jose State University College of Engineering

14. Typical microtome blades are manufactured from high carbon steel or heat-treated tool grade steel. Free From Impurities Anti-corrosive And Rust Resistant Profile of steel knives A: Strongly Plano Concave B: Plano Concave C: Wedge Shaped (Wedge Angle 27o) D: Plane Shaped (Wedge Angle 45o) Microtome Knife Material Selection San Jose State University College of Engineering

15. Depending on the application other materials are available for blade: Tungsten Carbide Glass Diamond Sapphire Disposable Blades (Stainless Steel) Sharpening Coarse sharpening Fine sharpening Stropping Performed less with the introduction of disposable knives More on Microtome Knives San Jose State University College of Engineering

16. Translational Guide Rails • Guide rails with V-grooves used to limit motion to1-DOF translation. • Forward Knife Motion • Vertical Raising And Lowering Of The Rigidly Held Sample San Jose State University College of Engineering

17. Previous Sliding Guides Replaced With Rigid And Play-free Cross-roller Linear Bearings • Reduces Wear On The Grooves • Relieves Stress In Bearings • Support More Load With Increasing Contact Surface • Roller bearings have less tendency to move out of position, or creep Pictures From References 6 and 7 San Jose State University College of Engineering

18. Digital Volumetric Imaging Normally • Microtome →Slices →Microscope Slide →Microscope →Viewing or Photography Digital Volumetric Imaging (DVI) • Microtome →Slices→Digital Photography→ Computer → 3-D Model • Precision in Both a Microtome System and a Microscopic Imaging System Picture From Reference 10 San Jose State University College of Engineering

19. Examples of DVI Datasets Pictures From Reference 10 San Jose State University College of Engineering

20. Focus and Color Images • CCD Chip → Grayscale Image • Color Filters • Capturing Images: Time-Dependent • Illumination and Motors → Heat → Expansion → Focus Issues San Jose State University College of Engineering

21. Solution to Expansion of Block • Add Refrigeration System • Add Auto Focus System • Objective Lens on Piezo San Jose State University College of Engineering

22. More Precise Motion • Higher Magnification Requires Thinner Sections. • Cross Roller Bearings → Air Bearing Slide • Stepper Motor & Linkages → Linear Motor San Jose State University College of Engineering

23. References • Steedman, H.F.; Section Cutting in Microscopy; Blackwell Scientific Publications Ltd, Oxford; England, 1960. • Leitz,Ernst; Leitz 1512/1516 rotary microtomes Instructions; Germany; 1984. • http://www.materials-sectioning.com • Woods and Ellis, Microtome: function and design http://home.primus.com.au/royellis/microt/microt.htm • http://biomicro.sdstate.edu/Hildrethm/Bio545/Techniques/Microtom.htm • http://www.schneeberger.com/ • http://www.mellesgriot.com/glossary/wordlist/glossarydetails.asp?wID=136 • http://www.theproductfinder.com/component/crorol.htm • Collins, Rodney; Personal Interview; San Francisco General Hospital, March 14, 2003 • http://www.meyerinst.com/html/resolve3d/default.htm San Jose State University College of Engineering

24. Thank You Very Much! Any Questions? San Jose State University College of Engineering